The kringle domain is a highly conserved structure found in five proteins involved in hemostasis: prothrombin, plasminogen, tissue plasminogen activator, urokinase and Factor XII. The exact function of the kringle domain remains uncertain, however, the two kringle domains in prothrombin likely play a role in the recognition of prothrombin as a substrate for the enzyme complex, Factor Xa and Factor Va (prothrombinase). The major goals of this project are to elucidate the role of prothrombin kringle domains in prothrombinase complex assembly on membrane surfaces, and to identify specific regions and amino acids within the kringle domain that define the interaction of prothrombin with Factor Xa and Factor Va. Prothrombin kringle deletion mutants have been constructed by oligonucleotide-directed loop-out mutagenesis. Prothrombin mutants in which the kringle domains are substituted for one another, and prothrombin chimera in which kringle domains are replaced by kringle domains from plasminogen or tissue plasminogen activator will be constructed as follows. The kringle domains of prothrombin will be selectively removed by loop-out mutagenesis and replaced by PCR generated kringle domains from prothrombin, plasminogen or tissue plasminogen activator. In addition, prothrombin mutants will be constructed with regional and point mutations in the kringle domains. The cDNA encoding these prothrombin mutants will be transfected into CHO cells, expressed, and the mutant proteins purified by immunoaffinity chromatography. To determine if the activity and functional specificity of these mutants has been altered, they will be evaluated for specific coagulant activity as well as for binding to, and activation by the Factor Xa/Factor Va (prothrombinase) complex on membrane surfaces. Mutants will be evaluated for direct binding to Factor Va in the presence and absence of Factor Xa, phospholipid and calcium ions, by measurement of fluorescence energy transfer. Their ability to serve as substrates for Factor Xa in the presence and absence of the cofactor, Factor Va, will be evaluated by fluorimetric kinetic analysis of DAPA binding to thrombin. Competitive inhibition of prothrombin activation will be assessed using thrombin- active-site-inhibited prothrombin kringle mutants. The results of these experiments will identify critical amino acids in the prothrombin kringle domain(s) which are involved in prothrombin binding to and activation by prothrombinase, and they will determine the functional role of kringle domains in conferring biological activity and specificity to blood coagulation proteins.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Physician Scientist Award (K11)
Project #
5K11HL002574-03
Application #
3087811
Study Section
Research Manpower Review Committee (MR)
Project Start
1991-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
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